VIROMER GREEN In vitro sirna/microrna Standard Transfection PRODUCT INFORMATION... 2 GENERAL... 2 BLUE OR GREEN?... 3 PROTOCOL GUIDELINES... 4 GENERAL REMARKS... 4 CELL CULTURE AND PLATING... 4 FORWARD/REVERSE TRANSFECTION: GENERAL WORKFLOW... 5 FORWARD TRANSFECTION PROTOCOL: 3-CONDITION OPTIMIZATION... 6 FINAL FORWARD TRANSFECTION PROTOCOL... 7 TO 100% KNOCK-DOWN DOSE-RESPONSE CURVES... 8 HIGH-THROUGHPUT SCREENING (HTS) APPLICATION DEVELOPMENT... 9 TROUBLESHOOTING / MINIMIZING BACKGROUND... 10 TECHNICAL SUPPORT AND ORDERS... 11 Viromer GREEN Manual 01/2016 1
PRODUCT INFORMATION General Technology: Viromer are polymeric transfection reagents of chemical nature taking advantage of a viral membrane fusion mechanism (hence their name). The membrane-like character is provided by alkyl moieties in combination with long chain fatty acids. During endocytosis, Viromer will become exposed to an acidic environment. The low ph renders the fatty acid moieties uncharged and hydrophobic, a switch that facilitates membrane crossing. This Active Endosome Escape technology maximizes transfection efficiency and reduces off-target effects. Key Benefits: Active Escape Technology Efficient for both adherent and suspension cells Zero Charge Compatible with serum or antibiotics. Gentle on cells Stable Particles Reproducible results Lipid free Reliable results, no interference with cell s lipid metabolism Reverse Transfection Ready for High-Throughput Screening Content: Viromer GREEN is available in 3 different formats Viromer GREEN 50 µl 300 µl VG-01LB-00 VG-01LB-01 Incl. Buffer GREEN (10 ml) Incl. Buffer GREEN (50 ml) 3 300 µl VG-01LB-03 Incl. Buffer GREEN (3 50 ml) In standard conditions (no optimization), 1 µl of Viromer GREEN is sufficient for 2 reactions of transfection in the 24-well plate format (incl. both target and control sirna). The number of transfections that can be performed will depend on the cell type, the optimal transfection scale and the culture plate format (see Table on Pages 6-7). Buffer GREEN (ph 7.2 aqueous solution) is required for diluting Viromer GREEN and the sirna or microrna needed to be transfected. Storage and use: Viromer GREEN should be stored at +2-8 C in the provided aluminum bag. It is then stable for 6 months (#VG-01LB-00) to 1 year (#VG-01LB-01/#VG-01LB-03). As the reagent is sensitive to atmospheric CO 2, it is recommended to always close the vial and tighten the cap immediately after use. Avoid contact of the reagent with dry ice. Viromer GREEN Manual 01/2016 2
Application: Viromer GREEN, as Viromer BLUE, is optimized for the transfection of sirna and microrna. For the transfection of plasmid DNA and mrna, please refer to Viromer RED and Viromer YELLOW. Quality control: Each batch of Viromer GREEN is tested for transfection using a PLK-1 and control sirna. Buffer GREEN was analyzed for composition, sterility and RNAse/DNase activity. MSDS are available at www.viromer-transfection.com. Product use limitations: This product is intended for research use only; it must not be used for therapeutic, veterinary or diagnostic applications. The purchase of this product implies a limited, nontransferable right to the purchaser to use this product, or parts from this product, only for its internal research. All further commercial applications of Lipocalyx products require a license from Lipocalyx GmbH. GREEN or BLUE? Viromer BLUE Viromer GREEN is a versatile standard with broad support in the user data. is more selective for particular cells. Viromer GREEN and Viromer BLUE are highly-effective on a wide range of standard and hard-totransfect cells including suspension cells, stem cells and primary cells without affecting cellular and lipid metabolism. Please refer to our selection guide at www.viromer-transfection.com If a cell type is not listed, parallel tests with both Viromer GREEN and Viromer BLUE are recommended. What is different? Viromer GREEN and Viromer BLUE differ in their surface and backbone chemistry but follow the same workflow. Why testing more than one? While we have optimized the Active Endosome Escape Technology, Viromer have no built-in cell specific motifs. Hence their uptake may differ between cell types and we cannot predict in advance which Viromer would be the best choice. Viromer GREEN Manual 01/2016 3
PROTOCOL GUIDELINES General Remarks Conditions of use and required materials: Warm all reagents to room temperature. The complexation and transfection of Viromer GREEN should be done under a sterile workbench using sterile, DNase/RNase free and apyrogenic tips and tubes. Complexes should be prepared freshly. Buffer GREEN (provided in the kit) is required to make dilutions of Viromer GREEN and sirna/microrna. Media: Viromer GREEN is fully compatible with all cell culture media, sera or antibiotics, so no dilutions or washings are required. The day before transfection, cells are seeded in complete medium which should be changed before starting (forward transfection). Forward/reverse transfection: Viromer GREEN can be used in forward or reverse transfection (page 5). For application in high-throughput screening (HTS), instructions are given at page 9. Cell Culture and Plating Grow cells to about 60-80% confluency. Use the volume of complete medium as mentioned in the table below. Recommended starting conditions for common cell culture plates are: Multiwell plate type 96 48 24 12 6 Adherent cells Cells seeded per well 12,000 30,000 60,000 125,000 250,000 Range * ±3,000 ±10,000 ±20,000 ±40,000 ±80,000 Suspension cells Cells seeded per well 48,000 120,000 240,000 500,000 1,000,000 Range * ±12,000 ±40,000 ±80,000 ±160,000 ±320,000 Medium per well (ml) 0.1 0.25 0.5 1 2 *In reverse transfection protocols, cell numbers should be on the higher end. Viromer GREEN Manual 01/2016 4
Forward/Reverse Transfection: General Workflow FORWARD TRANSFECTION REVERSE TRANSFECTION Viromer GREEN Manual 01/2016 5
Forward Transfection Protocol: 3-condition optimization Volumes given here support 24 or 96 well format. For 6 well, scale up 4 fold. 1. Dilute your target and control sirna/microrna to 2.8 μm using Buffer GREEN. For suspension cells, use 11 μm. Provide a volume of 15 μl per transfection >> Tube 1 2. Place a 3-μl droplet of Viromer GREEN onto the wall of a fresh tube. Immediately add 270 μl of Buffer GREEN and vortex for 3-5 s >> Tube 2 Always add Buffer GREEN to Viromer GREEN, not vice versa! 3. Complexation: Pipette 135 μl of the Viromer GREEN solution from Tube 2 onto the 15 μl of sirna/microrna solution in Tube 1. Mix swiftly and incubate for about 15 min at room temperature. Repeat with control sirna/microrna. 4. Add transfection complexes from step 3 to your cells. Titrate as per the table below to identify optimal conditions. Volume of complexes to add (μl per well) sirna/microrna on cells [nm] Transfection Scale 96 well 24 well 6 well Adherent Suspension Low 0.5 5 25 100 13 50 Standard 1.0 10 50 200 25 100 High 1.5 15 75 300 37 150 5 replicates 1 replicate 1 replicate 5. Incubate cells under their usual growth conditions. Monitor sirna/microrna effects 24-72 h after transfection. Viromer GREEN Manual 01/2016 6
Final Forward Transfection Protocol During optimization, a specific transfer volume and a specific transfection scale were identified. Please proceed with these specific settings and use the same workflow (step 1 to step 5) as for the optimization protocol for the final transfection protocol. The table below is a protocol using the 1.0 Transfection Scale (standard). All volumes are given for a single-well format. Please adjust all volumes according to the optimal transfection scale. 1. Start with diluting target and control sirna/microrna to 2.8 µm in Buffer GREEN (11 µm for suspension cells) 96 well 24 well 6 well comments 2. Viromer GREEN (μl) Buffer GREEN (μl) 1 90 1 90 4 360 Buffer GREEN onto Viromer GREEN Vortex 3-5s immediately 3. sirna/microrna 2.8 µm solution from step1 (μl) Viromer GREEN solution from step 2 (μl) 5 45 5 45 20 180 Mix swiftly and incubate for 15 min 4. Transfer volume (μl) 10 50 200 Incubate cells as usual replicates 5 1 1 5. Monitor sirna/microrna effects 24-72 h after transfection. Viromer GREEN Manual 01/2016 7
To 100% knock-down Dose-Response Curves As a final step in the optimization, the sirna/microrna concentration should be limited to the amount necessary to obtain a clear phenotype with maximum separation from any response to a control sirna/microrna (e.g. substantial knock-down). In dose-response experiments, a fixed amount of Viromer GREEN previously optimized and only vary the sirna/microrna concentration. The following is a standard protocol for adherent cells using 25nM sirna/mirna in the initial optimization step (1.0 Transfection Scale). Final sirna on cells [nm] 50 25 12.5 6 3 1. Dilute sirna/microrna in Buffer GREEN to μm.. Provide 5 μl of this solution. Use target and control sirna/mirna 5.5 2.7 1.3 0.7 0.35 2. a) Place 3 μl Viromer GREEN onto the wall of a fresh tube. b) Add immediately 270 μl of Buffer GREEN directly onto the Viromer GREEN droplet. Always add Buffer GREEN to Viromer GREEN, not vice versa! c) Vortex 3-5s. 3. Add 45 μl of the Viromer GREEN working solution (from step 2) onto the 5 μl of sirna/microrna solution (from step 1). Mix swiftly. Incubate for 15 min at room temperature. 4. 24-well plates: Transfect the cells using a transfer volume of 50-μl 96-well plates: Transfect the cells using a transfer volume of 10-μl (recommended to do at least 3 replicates) 5. Incubate and analyze knock-down of the target gene (24-72h post-transfection) Viromer GREEN Manual 01/2016 8
High-Throughput Screening (HTS) application development This section presents a typical HTS workflow for the standard conditions of the Viromer GREEN protocol (using 25 nm sirna/microrna with 50 µm Viromer GREEN). Based on a common reverse transfection protocol, all steps - complexation, transfer in wells and adding of cells - are performed at the same day following the sequence detailed below. NOTE: For Sensitive Cells: use 30% less Viromer GREEN, constant sirna/microrna. For Suspension Cells: constant Viromer GREEN, use 4-times amount of sirna/microrna. 1. Provide plates with sirna/microrna diluted in Buffer GREEN 96-well 384-well sirna [nm] 250 250 µl/well 10 5 2. Dilute Viromer GREEN to obtain 500 µm working solution. Use per plate: µl Viromer 9.8 18.8 µl Buffer GREEN 1090 2081 a) Place μl of Viromer GREEN onto the wall of a fresh tube. b) Add immediately Buffer GREEN onto the Viromer GREEN droplet. Always add Buffer GREEN to Viromer GREEN, not vice versa! c) Vortex for 3-5 s. 3. Dispense and mix the Viromer GREEN working solution and pipette it onto the pre-plated sirna. Incubate 15min for complex formation. µl/well 10 5 4. Add cells having a density of µl/well 96, adherent 180,000/mL 80 96, suspension 720,000/mL 80 384, adherent 90,000/mL 40 384, suspension 360,000/mL 40 NOTE: For phenotypic assays (lasting up to 3 days) plate only 1/3 of the abovementioned cell number. Seed a number of adherent cells sufficient for having a confluency of 90% at the end of the experiment. 5. Incubate cells as usual. Viromer GREEN Manual 01/2016 9
Troubleshooting / Minimizing Background The following steps are recommended for troubleshooting: 1. If transfection was successful but slightly toxic Change the medium 4h after transfection. 2. If there is still toxicity Try a different sirna/microrna. Keep in mind that even non-target control sequences may create background. 3. If there is still no signal Increase the incubation time before analysis of the targeted mrna or protein. The suggested time point of 24-72 hours is commonly accepted, but the half-life of a specific mrna or protein may be much longer. Parameters to adjust for minimizing background: Cell density: Test several seeding densities of the cells depending on growth rate and duration of the experiment. For adherent cells, target about 80% confluency at the time of knock-down analysis. In case of little to no effects on suspension cells, we recommend to increase the ratio [concentration of sirna:viromer complex] / [cell density]; either by reducing the cell density or by increasing the concentration of the sirna:viromer complex. Type of sirna: sirna designs have seen major updates to improve the specificity and reduce immunogenicity. We recommend using sirna pools and chemically modified sirnas. If immunogenicity is a concern, monitor levels of central genes coding for essential proteins, such as OAS1. For additional recommendations, please visit our support pages at www.viromer-transfection.com (incl. updated FAQs) or contact us! Viromer GREEN Manual 01/2016 10
TECHNICAL SUPPORT and ORDERS Technical Support Dr. Christian Reinsch christian.reinsch@lipocalyx.de +49 345 55 59 620 Info & Customer Service Bettina Weber bettina.weber@lipocalyx.de +49 345 55 59 625 Dr. Olivia Zabel olivia.zabel@lipocalyx.de +49 345 55 59 626 Dr. Sandra Lagauzère sandra.lagauzere@lipocalyx.de +49 345 55 59 663 Mail Orders order@lipocalyx.de FAX Orders +49 345 55 59 846 Webshop www.viromer-transfection.com Lipocalyx GmbH Weinbergweg 23 06120 Halle Germany Viromer GREEN Manual 01/2016 11